1. Field of the Invention
This invention relates to adenosine receptors from mammalian species and the genes corresponding to such receptors. Specifically, the invention relates to the isolation, cloning and sequencing of a novel adenosine receptor gene, termed A3. The invention also relates to the construction of eukaryotic expression vectors capable of expressing this novel adenosine receptor in cultures of transformed eukaryotic cells, and the production of the A3 adenosine receptor in such cultures. The invention relates to the use of such cultures of transformed eukaryotic cells to produce the A3 adenosine receptor for the characterization of novel and useful drugs.
2. Background of the Invention
Adenosine modulates diverse physiological functions including induction of sedation, vasodilatation, suppression of cardiac rate and contractility, inhibition of platelet aggregability, stimulation of gluconeogenesis and inhibition of lipolysis (see, Stiles, 1986, Trends Pharmacol. Sci. 7: 486-490; Williams, 1987, Ann. Rev. Pharmacol. Toxicol. 27: 315-345; Ramkumar et al., 1988, Prog. Drug. Res. 32: 195-247). Based on biochemical and pharmacological criteria, two subtypes of adenosine receptor have been differentiated (termed A1 and A2), which inhibit and stimulate adenylate cyclase, respectively (Stiles, ibid.; Williams, ibid. ). Substantial progress has been made concerning the biochemical and pharmacological properties of these adenosine receptors such as ligand binding characteristics, glycosylation, and regulation. Besides its effects on adenylate cyclase, adenosine has been shown to open potassium channels, reduce flux through calcium channels, and inhibit or stimulate phosphoinositide turnover through receptor-mediated mechanisms (see, Fredholm & Dunwiddie, 1988, Trends Pharmacol. Sci. 9: 130-134; Sebastiao et al., 1990, Br. J. Pharmacol. 100: 55-62; Stiles, 1990, Clin. Res. 38: 10-18; Nakahata et al., 1991, J. Neurochem. 57: 963-969). In addition, the A1 adenosine receptor has been purified to homogeneity from rat and bovine brain (Nakata, 1989, J. Biol. Chem. 264: 16545-16551; Olah et al., 1990, Arch. Biochem. Biophys. 283: 440-446).
Recently, the cDNAs that encode the A1 and A2 adenosine receptors have been cloned (see, Libert et al., 1989, Science 244: 569-72; Maenhaet et al., 1990, Biochem. Biophys. Res. Commun. 173: 1169-1178; Libert et al., 1991, EMBO J. 10: 1677-1682; Mahan et al., 1991, Molecular Pharmacol. 40: 1-7; Reppert et al., 1991, Molec. Endo. 5: 1037-1048). Molecular cloning of A1 and A2 receptors revealed they both belong to the superfamily of G-protein coupled receptors. Physiological and pharmacological studies, however, have suggested the existence of additional adenosine receptors besides A1 and A2 (Linet al., 1991, J. Biol. Chem. 266: 14457-14463; Ribeiro & Sebastiao, 1986, Prog. Neurobiol. 26: 179-209; Oliveira et al., 1991, J. Neurochem. 57: 1165-1171; Ali et al., 1990, J. Biol. Chem..265: 745-753).
The present inventors have recently obtained a number of G-protein related clones using a polymerase chain reaction (PCR)-based random cloning strategy (Zhou et al., 1990, Nature 347: 76-80). The present invention comprises a novel adenosine receptor gene, termed A3, the nucleotide sequence of this gene and the deduced amino acid sequence of its cognate protein, a method for determining the tissue distribution of expression of the gene, and the determination of a number of pharmacological characteristics of the A3 receptor protein. A related nucleotide sequence has been reported recently (see Meyerhof et al., 1991, FEBS Lett. 284: 155-160).